1. Field of the Invention
The present invention relates to a process for selectively esterifying .alpha.-L-aspartyl-L-phenylalanine to the corresponding alkyl esters using a proteolytic enzyme having specific esterase activity.
2. Description of the Prior Art
The esterification of .alpha.-L-aspartyl-L-phenylalanine by chemical means has been described in U.S. Pat. Nos. 3,933,781 and 4,173,562. A disadvantage of chemical methods is that esterification occurs at the aspartyl carboxyl group as well as at the phenylalanine carboxyl group so that undesirable by-products of formula I of chart A (hereinafter referred to as the diester) and formula II of chart A (hereinafter referred to as the aspartyl ester) are formed.
The diester and the aspartyl ester must be removed by purification steps in order to obtain the desired product.
Previous enzymatic esterification procedures have been directed to the esterification of single amino acids. They have not been concerned with the selective esterification problems that occur with dipeptides, particularly .alpha.-l-aspartyl-L-phenylalanine which has more than one carboxyl group.
For example, Ingalls, et al., BIOTECHNOLOGY AND BIOENGINEERING 17, 1627-1637 (1975) describes the esterification of a single amino acid, N-acetyltyrosine, using immobilized chymotrypsin or subtilisin Carlsberg in the free, unmodified form in a solvent system of water, ethanol and glycerol. A disadvantage of this method was that significant amounts of the glycerol ester of the amino acid were formed.
Klibanov, et al., BIOTECHNOLOGY AND BIOENGINEERING 19, 1351-1361 (1977) describes the esterification of a single amino acid, N-acetyltryptophan using immobilized chymotrypsin in a biphasic system of water and a water-immiscible solvent, chloroform.
An advantage of the present invention is that the carboxyl group of the phenylalanine moiety is selectively alkylated by enzymatic esterification in a single phase system to give .alpha.-L-aspartyl-L-phenylalanine alkyl ester without producing the undesireable diester and aspartyl ester. This is accomplished by contacting the dipeptide with an alcohol in the presence of a serine alkaline proteinase, an esterase exhibiting a preference for aromatic amino acids.